3-aminobutyric acid, or GABA, is the main inhibitory neurotransmitter in the mammalian brain. Dysfunctions in GABA-mediated inhibition have been implicated in the etiology of a variety of brain disorders. Furthermore, GABA receptors are targets for a host of therapeutic and endogenous modulators such as benzodiazepines, barbiturates, and neurosteroids. Key to understanding the etiology of these diseases and the molecular mechanisms of these modulators, is a detailed understanding of the activation mechanism of the GABA receptor. GABA, released from presynaptic terminals, binds to postsynaptic GABA receptors thereby gating an integral chloride pore. It is this chloride movement across the membrane that reduces the excitability of the postsynaptic neuron. The major focus of my laboratory has been to understand, at the molecular level, the mechanism of this activation process. Our efforts to date have been largely directed at identifying the components of the receptor that are fundamental in this activation process. For example, we have explored the structure of the binding site and identified amino acids that interact with the GABA molecule itself. Other investigations have been directed at locating the gate that keeps the channel closed and exploring the regions that control ion flux through the pore. The next step, and the primary aim of the present application, is to extend this static model of the receptor and begin to define the kinetics of the activation process. Last round, we introduced Voltage-Clamp Fluorometry to the ligand-gated ion channel field. This technique, using an environmentally-sensitive fluorophore attached to defined positions of the receptor, provides an ability to observe structural rearrangements in real time. Simultaneous two-electrode voltage clamp enables a correlation of these rearrangements with channel opening. This round, we will be employing this approach to ask questions about the movements of specific regions of the receptor and to elucidate the role of these movements in the activation process. We ultimately hope to understand the mechanism by which the binding of GABA in the inter-subunit binding cleft leads to the opening of the pore some 50 angstroms away and in the middle of the membrane. In addition to revealing the activation mechanism for these receptors, this information will be crucial for understanding the mechanism of the many GABA receptor modulators and, perhaps, provide information to facilitate the design of new neuroactive compounds that target and modulate GABA receptors in the brain. PUBLIC HEALTH RELEVANCE: GABA receptors are the key mediators of synaptic inhibition in the brain. This project seeks to understand, at the molecular level, how these GABA receptors work. This information is crucial for understanding the dysfunctions in GABA-mediated inhibition that have been implicated in many neural disorders (e.g. epilepsy, Parkinson's Disease, Down's Syndrome, and autism) as well as understanding, and developing new, compounds that target and modulate GABA receptors.